Supplementary Figures 1-5 from Single-Strand DNA-Binding Protein SSB1 Facilitates TERT Recruitment to Telomeres and Maintains Telomere G-Overhangs

Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

doi:10.1158/0008-5472.22408029.v1

00085472can142289-sup-135811_1_supp_0_nhrg8z.pdf (4.03 MB)

Supplementary Figures 1-5 from Single-Strand DNA-Binding Protein SSB1 Facilitates TERT Recruitment to Telomeres and Maintains Telomere G-Overhangs

journal contribution

posted on 2023-03-30, 23:30 authored by Raj K. Pandita, Tracy T. Chow, Durga Udayakumar, Amanda L. Bain, Liza Cubeddu, Clayton R. Hunt, Wei Shi, Nobuo Horikoshi, Yong Zhao, Woodring E. Wright, Kum Kum Khanna, Jerry W. Shay, Tej K. Pandita

Supplementary Figures 1-5. Supplementary Fig. 1. Analysis of single-strand G-overhang by native and denatured DNA gel electrophoresis. Supplementary Fig. 2. Telomere FISH analysis of chromosomal aberrations induced by Ssb1 depletion in mouse and human cells at metaphase. Supplementary Fig. 3. Analysis of hTERT and hSsb1 co-dependent association with telomeres by ChIP. Supplementary Fig. 4. Analysis of hSsb1 interaction with TRF1 and TRF2 by co-immunoprecipitation and ChIP analysis of their influence on hSsb1 recruitment to telomeres. Supplementary Fig. 5. Measurement of in vitro telomerase activity (TRAP assay) in control and hSsb1 depleted cells.

History

ARTICLE ABSTRACT

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1–TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858–69. ©2015 AACR.